A Global View of Translation in Vesicular Stomatitis Virus Infected Cells
Neidermyer Jr, William J.
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AbstractInfection of mammalian cells with vesicular stomatitis virus (VSV) results in the inhibition of cellular translation, while viral translation proceeds efficiently. This host “shutoff” is mediated by multiple mechanisms. VSV infection alters the phosphorylation of cellular eIF4E-BP1, thereby sequestering the cap-binding protein eIF4E. The viral matrix protein, M, blocks nuclear export of host mRNPs, and inhibits cellular transcription, suppressing the host mRNA pool. VSV replicates within the cytoplasm, and during transcription the viral polymerase transcribes 5 mRNAs that are structurally identical to cellular mRNAs with respect to their 5' cap-structure and 3'-polyadenylate tail. We employed the global approach of massively parallel sequencing cytoplasmic, monosome, and polysome-associated mRNA to interrogate the impact of VSV infection of HeLa cells on translation. Analysis of sequencing reads in the different fractions shows > 60% of the cytoplasmic and polysome-associated reads map to the 5 viral mRNAs by 6 hours post-infection (hpi), which corresponds with host shutoff. Consistent with the overwhelming abundance of viral mRNA on polysomes, reads mapping to cellular genes were reduced after infection, although a subset exhibited increased polysome association. Analysis of viral and cellular mRNA distributions within polysome profiles by quantitative PCR supports a redistribution of cellular mRNAs to smaller polysomes and monosomes in infected cells. To test the contribution of blocking nuclear mRNA export to shutoff, we infected cells with a viral mutant in M (M51R), which is defective in blocking nuclear export. This analysis reveals > 50% of cytoplasmic and polysome-associated reads map to viral mRNAs by 6 hpi, confirming the role of viral mRNA abundance in host shutoff. Furthermore, interferons were differentially expressed 6 hpi with M51R. Interferons were not upregulated at 2 hpi, or during infections with VSV harboring wild-type M, indicating viral replication is required for innate immune sensing, and that blocking nuclear export principally inhibits interferon gene expression. These results provide a global view of host mRNA translation in response to VSV, supporting a model in which viral mRNA abundance is a key determinant of host shutoff, with inhibiting mRNP export and eIF4E sequestration contributing additional effects.
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